CMV is a ubiquitous Herpes-type virus having a linear, double-stranded DNA genome of about 236,000 kbp (Gibson, “Structure and Formation of the Cytomegalovirus Virion,” Human Cytomegalovirus, Curr. Topics in Microbiol. Immunol. 325: 187-204, Shenk and Stinski, eds., Springer-Verlag Berlin Heidelberg (2008)). The CMV genome is organized as two regions of unique sequences, unique long (UL) and unique short (US) (Kotenko et al., PNAS USA 97(4): 1695-1700 (Feb. 15, 2000)). The UL region is flanked by a set of inverted repeats, terminal repeat long (TRL) and internal repeat long (IRL; inverted repeat of TRL), and the US region is flanked by another set of inverted repeats, internal repeat short (IRS; inverted repeat of TRS) and TRS (terminal repeat short) (Kotenko et al. (2000), supra; see, also, Dunn et al., PNAS 100(24): 14223-14228 (Nov. 25, 2003)).
CMV infects 40-80% of humans before puberty. CMV becomes latent after primary infection, which often is asymptomatic. Even recurrent infection is asymptomatic in most cases or leads to only mild disease in an immunocompetent host. However, in congenitally infected infants and immunocompromised patients, such as allograft recipients (Meyers et al., J. Infect. Dis. 153: 478-488 (1986)) or autoimmune deficiency syndrome (AIDS) patients (Drew, J. Infect. Dis. 158: 449-456 (1988); and Drew, Clin. Infect. Dis. 14: 608-615 (1992)), CMV can cause severe and sometimes life-threatening diseases, including retinitis, gastrointestinal disorders (see, e.g., acute gastric mucosal lesion (AGML) as reported by Matsui et al., Internal Medicine 49: 1265-1267 (2010)), and encephalitis (Drew (1992), supra). Half of allogeneic stem cell recipients develop CMV infection in the first 100 days after transplantation (de la Cruz-Vicente et al., Transplant Proc. 42(8): 3230-3231 (October 2010)). CMV end-organ disease is a serious, frequent complication of allogeneic stem cell transplantation (de la Cruz-Vicente et al., Transplant Proc. 42(8): 3228-3229 (October 2010)).
Early administration of antiviral drugs (e.g., ganciclovir and foscarnet) can have significant beneficial effects on the prognosis of a patient (Jahn et al., Intervirology 35: 60-72 (1993); Schmidt et al., N. Engl. J. Med. 324: 1005-1011 (1991)). Since clinically effective antiviral therapy is available, early and sensitive diagnosis is significantly important.
Various methods have been used to assay CMV. Anti-CMV antibodies, in particular IgM antibodies, can be used as a marker for CMV infection. However, the detection of anti-CMV antibodies has limited value in the discrimination of latent and active infection. Naumnik et al. (Transplant Proc. 39(9): 2748-2750 (November 2007)) have reported that detection of CMV-IgM antibodies by various immunoassays is not sensitive enough for diagnosis and cannot be used for monitoring during the active period in renal transplant recipients.
Most currently employed viral detection methods do not unambiguously allow for prediction of whether a given infection will become symptomatic. Serological methods are indirect and often lack sensitivity. While viral culture is a more direct diagnostic parameter for CMV viremia, such as viral culture from blood cells, the method is technically difficult and does not enable rapid diagnosis. Moreover, viral culture does not necessarily correspond to CMV disease. Isolation of virus from peripheral leukocytes may not predict clinical symptoms in some immunosuppressed patients (Delgado et al., J. Clin. Microbiol. 30: 1876-1878 (1992)). Similarly, urinary or pharyngeal shedding of the virus frequently occurs without clinical symptoms and organ involvement. Amplification of CMV DNA in peripheral leukocytes using PCR can occasionally detect CMV DNA, such as latent viral genomes, in peripheral leukocytes without CMV-related diseases (Jahn et al. (1993), supra; Zipeto et al., J. Clin. Microbiol. 30: 527-530 (1992); and Delgado et al. (1992), supra). The antigenemia assay has been frequently used for the early diagnosis of acute symptomatic CMV infection.
The antigenemia assay involves the detection of the structural protein pp 65 using specific antibodies (Storch et al., J. Clin. Microbiol. 32: 997-1003 (1994); Gerna et al., J. Infect. Dis. 164: 488-498 (1991); and Gerna et al., J. Clin. Microbiol. 30: 1232-1237 (1992)). However, the number of pp 65-positive cells may be very low early in infection, and the stability of the pp 65 protein appears to be limited (Chou et al., Curr. Opin. Infect. Dis. 5: 427-432 (1991)). In addition, the antigenemia assay requires immediate sample processing and suffers from non-standardization of sample processing between laboratories, inaccurate reflection of the viral load in the blood compartment, occasional negative results in the presence of CMV disease, a time-consuming and laborious procedure, an inability to be automated, a lack of feasibility during periods of neutropenia, and a lack of objectivity of quantification (Choi et al. (2009), supra; Gimeno et al., J. Clin. Microbiol. 46(1): 3311-3318 (October 2008; epub Aug. 27, 2008)).
Since viral replication requires transcription, the presence of CMV mRNA as a marker of active CMV infection has been investigated (Bitsch et al., J. Infect. Dis. 167: 740-743 (1993)). RNA amplification has been used (Bitsch et al. (1993), supra; Meyer et al., Mol. Cell. Probes 8: 261-271 (1994); and Gerna et al. (1992), supra). Sillekens et al. (U.S. Pat. No. 6,306,602 B1, EP 0 729 518 B1, EP 0 973 948 B1, CA 2 172 142, and CA 2 281 793) discloses that the detection of a certain late mRNA, namely the matrix tegument protein pp 67 encoding gene sequence (UL65), which is expressed during the late phase of CMV infection, is related to the appearance of clinical symptoms of CMV disease. Analysis of immediate-early 1 (IE1) and immediate-early 2 (IE2) transcripts (Loh et al., Acta Virol. 53(4): 261-269 (2009)) and R-gene transcripts (CMV R-gene™ kit (Argene, Inc., Shirley, N.Y.); Marque-Juillet et al., Pathol. Biol. (Paris) 58(2): 162-165 (April 2010; epub 10.24.09)) also have been described. The CMV HHV6,7,8 R-gene™ kit of Argene, Inc. (Shirley, N.Y.), reportedly enables the detection of ppUL83 transcripts for CMV, along with U57 transcripts for HHV-6, U42 transcripts for HHV-7, and ORF26 transcripts for HHV-8 (see, also, Raggam et al., Med. Microbiol. Immunol. 199(4): 311-316 (November 2010; epub 6.18.10). A PCR-ELISA method employing primers for glycoprotein H (gH5) has been described by Allen et al., J. Clin. Microbiol. 33(3): 725-728 (March 1995)).
While real-time PCR is widely considered to be an efficient and highly sensitive technique for the evaluation of CMV DNA kinetics (Hong et al., Clin. Chem. 50: 846-856 (2004); Kearns et al., J. Clin. Microbiol. 39: 2364-2365 (2001); Piiparinen et al., J. Clin. Virol. 30: 258-266 (2004); and van Doornum et al., J. Clin. Microbiol. 41: 576-580 (2003)), the sensitivity and reliability of CMV DNA detection is greatly dependent on target sequence and primer selection, since there is sequence variation among strains of CMV throughout the genome (see, e.g., Caliendo et al., J. Clin. Microbiol. 38: 2122-2127 (2000); and Gault et al., J. Clin. Microbiol. 39: 772-775 (2001)). Chou (J. Clin. Microbiol. 30(9): 2307-2310 (September 1992)) compared the immediate-early region exon 4 sequence of six clinical CMV strains with two laboratory strains and found 8.1% inter-strain variation at the peptide level and 18% inter-strain variation at the nucleotide level in 407 codons in exon 4. The variation reportedly occurred sporadically throughout the exon without apparent strain grouping. Chou et al. also reported that some of the published oligonucleotide primers proposed for amplification of exon 4 sequences in the diagnostic detection of CMV by PCR showed sequence mismatches with the examined strains. Such mismatch reportedly reduced amplification sensitivity by up to 100-fold.
Numerous CMV gene targets have been used for PCR amplification and quantitation. Examples include the HindIII X region (Wolff et al., J. Molec. Diag. 11(2): 87-92 (2009)), UL 117 (Wolff et al. (2009), supra), UL123 (Wolff et al. (2009, supra); Ducroux et al., J. Clin. Microbiol. 46(6): 2078-2080 (2008); and Gimeno et al., J. Clin. Micro. 46(10): 3311-3318 (2008)), UL125 (Boeckh et al., J. Clin Micro. 42(3): 1142-1148 (2004)), UL126 (Boeckh et al. (2004), supra), UL54 (Wolff et al. (2009), supra; Fukushima et al., J. Virol. Meth. 151: 55-60 (2008); Sanghavi et al., J. Clin. Virol. 42: 335-342 (2008); and Hantz et al., Antiviral Res. 81: 64-67 (2009)), UL55 (Wolff et al. (2009), supra; Fukushima et al. (2008), supra; Bourne et al., Virol. 382: 28-36 (2008); and Pang et al., J. Clin. Micro. 46(12): 4004-4010 (2008)), UL55/UL123 (Boeckh et al. (2004), supra; and Limaye et al., JAMA 300(4): 413-422 (2008)), UL73 (Bourne et al. (2008), supra), UL74 (Bourne et al. (2008), supra), UL75 (Fukushima et al. (2008), supra), UL83 (Wolff et al. (2009), supra; Ducroux et al. (2008), supra; and Fukushima et al. (2008), supra), and US17 (Sanghavi et al. (2008), supra).
Habbal et al. (J. Med. Microbiol. 58: 878-883 (2009)) describe the comparative sensitivity of published primers for single-round, real-time PCR assay of CMV. Three primer sets located in the glycoprotein B (UL55) gene region were found to be the most sensitive in the analysis of CMV strain AD169; however, two of the three primer sets showed a considerable number of mismatches with clinical isolates in a BLAST search. Two other pairs of primers from the lower matrix phosphoprotein (UL83) gene and the DNA polymerase (UL54) gene showed reasonable sensitivity and no mismatches with clinical isolates. Further testing indicated that the UL55 primer set, which did not show a considerable number of mismatches with clinical isolates by BLAST searching, and the UL54 primer set were the most sensitive. Additionally, the analytical sensitivity of the PCR inversely correlated with the size of the PCR product. Habbal et al. suggest a primer pair for the UL55 gene as a candidate for a standardized PCR for CMV.
Mendez et al. (J. Clin. Microbiol. 36(2): 526-530 (February 1998)) evaluated PCR primers for early diagnosis of CMV infection following liver transplantation. Peripheral blood leukocytes (PBLs) and serum were assayed. Primer pairs directed to the HindIII-X fragment region of CMV reportedly detected target DNA with 94% sensitivity, compared to 87% sensitivity with primer pairs directed to an EcoRI fragment D, 32% sensitivity with primer pairs directed to the immediate-early antigen 1 (IEA1) gene, and 20% sensitivity with primer pairs directed to the major immediate-early (MIE) gene. The sensitivity of the primers for amplifying CMV DNA associated with symptomatic infection reportedly ranged from 100% (HindIII-X) to 20% (MIE); specificity reportedly was inversely related to sensitivity (45% for HindIII-X and 91% for MIE). CMV DNA from PBLs reportedly was a more sensitive target for HindIII-X and EcoRI-D primer sets. Overall, primers directed to the HindIII-X fragment region were found to be optimal for early detection of CMV DNA in PBLs and sera from symptomatic liver transplant recipients.
Boeckh et al. (J. Clin. Microbiol. 42(3): 1142-1148 (March 2004)) compared PCR assay of plasma using three different primer sets (UL125 alone, UL126 alone, and UL55/UL123-exon 4) with pp 65 antigenemia assay and blood culture. Boeckh et al. reported that plasma PCR detected CMV more frequently in blood specimens that either the antigenemia assay or blood cultures. The PCR assay employing UL55/UL123-exon 4 primers reportedly performed better than the PCR assay employing primers for either UL125 or UL126 with regard to sensitivity, specificity and predictive value when compared to the antigenemia assay.
Gimeno et al. (J. Clin. Microbiol. 46(10): 3311-3318 (October 2008; epub 8.27.08) compared an automated real-time PCR assay (Cytomegalovirus PCR Kit, Abbott Molecular, Inc., Des Plaines, Ill.) with the antigenemia assay in the surveillance of active CMV infection in 42 allogeneic hematopoietic stem cell transplantation recipients. Gimeno et al. found that the real-time PCR assay allowed an earlier diagnosis of CMV from the blood. Analysis of the kinetics of DNAemia levels at a median of seven days post-treatment reportedly allowed the prediction of the response to CMV therapy. The PCR assay reportedly tested positive before the onset of symptoms and during the disease period for two patients that developed CMV colitis.
The use of three different primer sets (IE, LA, and gB) in PCR assay of viral isolates and the use of gB primers in PCR assay of urine and dried blood spots (DBS) on Guthrie Cards also has been assessed (Distefano et al., BMC Pediatr. 4: 11 (June 2004)). Primers directed to the gB fragment region reportedly were the best choice for the PCR detection of CMV DNA in positive isolates.
The AMPLICOR CMV PCR test (Roche Molecular Systems, Inc., Branchburg, N.J.) employs primers that target a region of the polymerase gene (UL54). The test reportedly is specific and sensitive and has a limit of detection adequate to detect CMV in cerebrospinal fluid (CSF) of 95% of patients with neurological CMV disease (Weinberg et al., J. Clin. Microbiol. 36(11): 3382-3384 (November 1998)).
The region of the polymerase gene has been suggested to be a better choice than the major immediate early (MIE) region for primer selection (Wirgart et al., J. Clin. Microbiol. 36(12): 3662-3669 (December 1998); see, also, Thorne et al., Diagn. Mol. Pathol. 16(2): 73-80 (June 2007)). However, Leruez-VIIIe et al. (J. Clin. Microbiol. 46(4): 1571-1572 (April 2008)) have reported that real-time PCR employing primers directed to a strictly conserved sequence in exon 4 of MIE was much more sensitive than the pp65 antigenemia assay, and compared favorably to the CMV R-gene real-time PCR (Argene, France), except at DNA load levels near the threshold values of the two techniques.
The Qiagen RealArt CMV LightCycler PCR (Qiagen, Germantown, Md.), which employs primers that target the CMV MIE region, was compared to the Roche CMV UL54 Analyte-Specific Reagent PCR (Roche Molecular Systems, Inc., Indianapolis, Ind.), which employs primers that target UL54, and the Digene Hybrid Capture System CMV DNA test (version 2.0; Digene Corp., Gaithersburg, Md.), which is a solution hybridization antibody capture assay for the chemiluminescent detection and quantitation of CMV DNA in leukocytes, by Hanson et al. (J. Clin. Microbiol. 45(6): 1972-1973 (June 2007)) using whole virus standards and plasma specimens from allogeneic stem cell transplant recipients. The Qiagen and Roche tests reportedly were more sensitive than the Digene test, detected CMV DNA earlier after transplant, and remained positive longer once antiviral treatment was initiated.
Koidl et al. (J. Virol. Methods 154(1-2): 210-212 (December 2008); epub 10.1.08) evaluated assay of CMV DNA in whole blood preserved with EDTA and plasma preserved with EDTA using two commercially available methods of detection, i.e., CMV HHV6,7,8 R-gene™ kit of Argene, Inc. (Shirley, N.Y.) and the artus CMV LC PCR kit (Qiagen, Hamburg, Germany). Whole blood preserved with EDTA was found to be superior to plasma preserved with EDTA when using the CMV HH6,7,8 R-gene™ kit.
As reported by Habbal et al. ((2009), supra), several evaluation studies have demonstrated significant differences in sensitivity of various in-house (also referred to as home-brew) and commercial PCRs (see, e.g., Allen et al. (1995), supra; Distefano et al. (2004), supra; Weinberg et al. (1998), supra; and Wirgart (1998), supra). Moreover, the most frequently used primers have failed to detect CMV in some isolates (see, e.g., Allen et al. (1995), supra; Distefano et al. (2004), supra; and Wirgart et al. (1998), supra). Habbal et al. concludes that such observations imply that the most appropriate target region for amplification has not yet been defined, and that standardization is still required for reliable and comparable PCR results. Further, as highlighted by Gimeno et al. ((2008), supra) and arguably supported by Koidl et al. ((2008), supra), analytical performance is also impacted by methods of detection and extraction. In this regard, Xue et al. (Clin. Chem. Lab Med. 47(2): 177-181 (2009)) concluded PCR should be used in combination with the antigenemia test to monitor CMV infection and predict its outcome, at least in the context of renal transplantation. Gimeno et al. ((2008), supra) on the other hand, opines that real-time PCR displays several advantages over the antigenemia assay for monitoring active CMV infection in allogeneic stem cell transplant patients. Such advantages include earlier diagnosis of active CMV infection, more reliable marker of successful clearance of CMV from blood, and the possible prediction of the response to CMV treatment by analysis of kinetics of CMV DNAemia at the time of initiation of pre-emptive therapy or early thereafter.
In view of the foregoing, there remains a need for oligonucleotide primers and probes for the amplification and subsequent detection of CMV that are specific, sensitive, and have a limit of detection adequate to detect CMV in any biological fluid sample, in particular blood or plasma. Desirably, the primers and probes enable detection of most strains of CMV, and even more desirably, all strains of CMV. The present disclosure seeks to provide further oligonucleotide primers and probes that, when used in combination in a PCR assay of CMV, provide a highly robust assay that is not only specific and sensitive but highly reproducible. This and other objects and advantages, as well as inventive features, will become apparent from the detailed description provided herein.